62
International Journal of Biotechnology for Wellness Industries, 2014, 3, 62-68
Bioproduction of Conjugated Linoleic Acid in Yogurt by Probiotic
Bacteria
Kianoush Khosravi-Darani1,*, Fatemeh Seyed Reihani2 and Reza Feili2
1
Research Department of Food Technology, National Nutrition and Food Technology Research Institute,
Faculty of Nutrition Sciences and Food Technology, Shahid Beheshti University of Medical Sciences, P. O.
Box 19395-4741, Tehran, Iran,
2
Food Technology Division, School of Industrial Technology, Universiti Sains Malaysia, 11800 Minden,
Penang, Malaysia
Abstract: Conjugated linoleic acid as unique fatty acid of milk fat has beneficial properties including antioxidant‚
anticarcinogen‚ antidiabetic‚ antiblood pressure‚ stimulating the body immune system and reducing cholesterol. The aim
of this study was assessing ability of some probiotics for biotransformation of linoleic acid to conjugated linoleic acid.
Effect of process variables were investigated on production of conjugated linoleic acid in probiotic yogurt. An 8 run
Plackett-Burman design was used to study the effect of 7 variables included: addition of supplements (whey powder‚ the
amount of added grape seed oil), fermentation (temperature, pH, incubation time) and inoculum condition (age and size)
on biomass and conjugated linoleic acid production in probiotic yogurt containing strains of Lactobacillus acidophilus
La5, Bifidobacterium bifidum and Propionibacterium freudenerchii grown in medium containing free linoleic acid. The
highest amount of conjugated linoleic acid was obtained by addition of 8% w/v whey powder, addition of 4%v/v grape
seed oil in pH=6.0, inoculation of 0.7%v/v Inoculum of 36 h age and fermentation at temperature of 35°C for 27 h. This
research showed that at the optimized conditions‚ the amount of conjugated linoleic acid in probiotic yogurt was
increased by 40% from an average of 8.01 mg/g fat in non-treated yogurt to 11.03 mg/g fat of probiotic yogurt containing
grape seed oil.
Keywords: Probiotic yogurt‚ Conjugated linoleic acid‚ Lactobacillus acidophilus La5, Bifidobacterium bifidum,
Propionibacterium freudenerchii, Plackett-Burman Design.
1. INTRODUCTION
Conjugated linoleic acid (CLA) is a geometrical
isomer of linoleic acid which can be found naturally in
meat and milk fat [1]. Cis-9 transe-11 isomer of CLA is
the most biologically active and dominant isomer in
milk fat which include 75-90% of the total CLA isomers
in milk fat [2]. Studies on the effect of omega-6 fatty
acids on human health started in 1998. Numerous
health-promoting properties associated with particular
isomers of CLA are reported [3, 4]. This unique fatty
acid shows numerous health benefits, including
antiatherogenic [5, 6], antidiabetic [3], antiinflammatory, anticarcinogenic and antiobesity [7, 8]
properties antioxidant‚ adjusting the body immune
system [9]‚ anti-hyper blood pressure‚ cholesterol
reducing, and reduction of body fat [10, 11]. These
anticarcinogenic effects of CLA e.g. mammary [12] and
prostate tumors [13], as well as inhibitor of skin
papillomas [4], colon aberrant crypt foci [14], and
metastasis of human breast cancer cells in animal
models [15] are attributed either to mixtures of CLA
isomers or to the pure c9, t11 isomer [16]. Kinetics of
*Address correspondence to this author at the Research Department of Food
Technology, National Nutrition and Food Technology Research Institute,
Faculty of Nutrition Sciences and Food Technology, Shahid Beheshti
University of Medical Sciences, P. O. Box 19395-4741, Tehran, Iran; Tel: +9821-22086348; Fax: +98-21-22376473; E-mail: [email protected]
ISSN: 1927-3037/14
biohydrogenation in Lactobacillus acidophilus
production of CLA has been also studied [17].
for
Microbial production of CLA can be achieved by
intestinal bacteria. Fermentative production of CLA in
milk was firstly reported in 1989 and 1990 [18, 19].
Then several research was conducted on the impact of
some process variables on CLA production including
incorporation of non- fat dry milk, temperature [20],
presence of LA in different media [21], reaching to
acidic pH [22] whey incorporation, LA and
oligosaccharides addition [23], time and microorganism
e.g. probiotic strain of Lactobacilli, Bifidobacteria,
Propionibacteria,
Leuconostoc,
Lactococcus,
Enterococcus, and Pediococcus [24-28]. Also effect of
process variables e.g. air, additives, and pH, addition of
prebiotics [29] and castor oil, (as a rich source of in the
triacylglycerol form of ricinoleic acid) [30]. Rapid
detection of CLA producing bacteria was reported by
spectrophotometric [31].
Our previous study showed that the highest amount
of CLA was obtained by addition of 4% whey powder,
addition of grape seed oil in pH=6.0‚ amount of added
oil was 4%v/v milk, incubation temperature of 35˚C and
termination of incubation at pH=4.8 [30, 31]. The
amount of CLA in probiotic yogurt increased from 7.07
mg/g in non-treated yogurt to 9.23 mg/g fat.
© 2014 Lifescience Global
Bioproduction of Conjugated Linoleic Acid in Yogurt
International Journal of Biotechnology for Wellness Industries, 2014, Vol. 3, No. 2
In the present study, three probiotic strains
Lactobacillus acidophilus La5, Bifidobacterium bifidum
and Propionibacterium freudenerchii were compared
from the view of CLA production yield. Then the effect
of process variables on production yield was
investigated separately by Plackett-Burman design
(PBD) using a mixed culture of selected probiotic.
5
4
3
Effects
2
1
0
1
2
3
4
5
6
7
Variables
Figure 1: Impact of different variables on CLA production in
Plackett-Burman design, (1: Inoculum age, 2:whey, 3: time,
4:oil, 5: Inoculum size, 6: Temperature, 7:pH).
Figure 2: Sensory evaluation of yogurt with and without
grape seed oil (GSO) in terms of flavor, taste and texture.
2. MATERIALS AND METHODS
2.1. Sample and Microorganism Preparation
Reconstituted milk made of 10% milk solid non fat
was pasteurized for 30 min at 90°, and cooled to 35°
C to prevent possible heat shock to probiotic bacteria.
6
Inoculum size of 10 probiotic was added into the
reconstituted milk and was slowly stirred by magnetic
stirrer for 15 min till it dissolved completely. Packs of
starter cultures which were supplied by Chr-Hansen
63
(Horsholm, Denmark) were kept in a -18 °C freezer.
This process was performed alongside the flame and
under sterile conditions. An amount of 0.4% or 0.8 % of
inoculums was added to the milk to produce probiotic
yogurt. The dilution degree of powder dissolved in milk
was as the recommended amount stated on the
package of Christian Hansen (equivalent to 0.000048).
Samples were prepared by addition of starter culture
including the probiotic bacteria into milk with 3% fat [20,
21]. Whey powder and grape seed oil was purchased
from Gela dairy product Industry (Amol, Iran) and
Azran-sanat (Esfahan, Iran)
Samples were checked every half an hour and their
pH reduction was monitored according to preliminary
experimental design. At this stage, grape seed oil was
added to the samples. The amount of addition was 0.1
% of the volume of the inoculated milk. After the
incubation according to experimental design, produced
probiotic yogurt samples placed inside a container full
of ice. Thereafter, they were transferred to the freezer
and subjected to approximately 0 °C. Consequently
after about 30 min incubation temperature decreased
to 20°C to prevent bacterial activity and further
decrease in pH. Samples were allowed to remain at
this condition for about two hours to ensure the
temperature is fixed around 4 °. It should be noted
that probiotic bacterial inoculums were not used for
subsequent experiments in order to prevent and reduce
the loss of viability of them. After measuring the CLA
content of probiotic yogurt production, sample with the
highest amount of CLA content was separated for
further 8 final main tests including evaluation of the
CLA content immediately after production and also
after one week storage of probiotic yogurt, measuring
yogurt fat, non-fat dry matter, pH, acidity (in terms of
lactic acid bacteria), probiotic bacteria counts, and
sensory evaluation were performed on the final product
(each test was performed in triplicates) [20, 21, 29, 32,
33].
2.2. Chemical Analysis
Extraction and separation of CLA were conducted
according to the method used by Lin et al. [34, 35]. A
2.5 gram of yogurt sample was mixed with 10 mL of
chloroform-methanol in a 2:1 volume ratio (V/V) and
was centrifuged for 3 min at 2800 rpm and the resulting
mixture was centrifuged again for 20 min at -8 ° in
4000 rpm. Thereafter, three phases composed of three
layers was specified including the surface (blue),
middle (yogurt dry matter) and the lower organic layer
containing the fatty acids associated with chloroform
64
International Journal of Biotechnology for Wellness Industries, 2014, Vol. 3, No. 2
Khosravi-Darani et al.
and methanol. The bottom layer was removed by
Pasteur pipette and after addition of 0.3g of sodium
sulfate was kept inside the refrigerator overnight. This
phase consisted of three new phases; top layer which
was remained from the previous aqueous phase; the
middle layer which was a mixture of chloroformmethanol with a mixture of fatty acids, and last layer
which consisted of sodium sulfate. At this stage, the
supernatant was removed by using Pastor Pipette. The
middle phase was separated from sodium sulfate by
performing decantation and then used in our
experiment [20, 21, 29].
bifidum were tested for their ability to convert linoleic
acid to conjugated linoleic acid. Growth and CLA
production were followed during incubation for 37 h in
reconstituted skim milk containing 0.2% addition of
safflower oil in pH=6.0‚ and amount of added oil was
1.4 ml/l milk. MRS-bile agar medium was used for
selective count for probiotic. 0.15% bile integral part
was added to the basal medium which is MRS-agar
medium. Therefore, growth of traditional bacterial
culture was prevented in yogurt. Incubation in aerobic
conditions at 35 ° C for 72 hours led to absolute growth
of L. acidophilus.
To remove the mixture of organic solvents
(chloroform-methanol) rotary evaporator was used and
all the solvents of fatty acids were completely dried off.
At the end, what remained was only a mixture of fatty
acids. Thereafter, in order to saponify fatty acids 1 mL
solution of 1N sodium hydroxide in methanol was
added into the solution and then it was left in the COD
reactor for 15 min at 100 °. After cooling it back to the
room temperature 6 mL of 4% hydrochloric acid
solution in methanol was added to the solution in order
to methylate the fatty acids and then incubated for 20
min in water bath at 60 °C.
2.5. Statistical Methods
2.3. Methyl Ester Forming for Gas Chromatography
Analysis
At this stage, 2 mL deionized water was added to
the sample and homogenized for 15 min by using a
vortex mixer. As a result, polar bonds between
methanol and water caused release of methyl ester.
Then 5mL N-hexane was added and the sample was
homogenized for 15 min by using a vortex mixer. This
caused transfer of methyl ester from aqueous to
organic phase. The organic phase was washed out
twice and aqueous phase was completely removed.
Finally, one gram of anhydrous sodium sulfate was
added to the organic phase and 1 l of this sample was
injected in gas chromatographic columns (Capillary
BP10) in a philips scientific model 4410 gas
chromatography (UK). Column used was 25 m in
length and 0.22 mL in diameter; and with a thickness of
0.25 micro liters. The initial temperature of column was
150°C with 1 min holding time, injection temperature at
250°C, the final temperature 230°C and 10 min
duration, with a temperature ramp of 5°C in 1 min.
Plackett-Burman experimental design was used to
study the effect of 7 variables in 8 treatment
combinations as Table 1 shows. PBD is one of the
highly fractional designs which allows for the study of k
= (N-1)/ (L-1) factors, each with L levels with N
experimental trials. The usefulness of the design lies in
the fact that in determining the effects of one variable,
the net effects of changing other variables cancel out
so that the effect of each variable on the system can be
independently determined. As it is known, in the PBD
only two levels can be considered for each factor.
Table 1 shows the factors that include some medium
components, environmental factors, and inoculum
condition. The corresponding levels in this table were
chosen on the basis of the preliminary tests. Table 2
shows selected experimental factors and a PBD for
conducting eight experimental trials. All the trials were
done in triplicate. The elements, + (high level) and (low level) represent the two different levels of the
independent factors examined. The most important
result obtained from this design is screening of 7
variables and identification of the most important ones
CLA production in probiotic yogurt.
Table 1: Seven Variables and Their Levels selected for
Evaluation of Their Impact by Plackett-Burman
Design on CLA Production
Variables
Lower level(-)
Higher level (+)
A:
Whey powder (% w/v)
2
4
B:
oil % (v/v)
2
4
C:
pH
4.5
6
D:
Time (h)
18
27
2.4. Microbiological Methods
E:
Seed size (% v/v)
0.4
0.8
Probiotic strain including L. acidophilus La5,
Propionibacterum
freudenreichii,
Bifidobacterium
F:
Temperature (˚C)
35
39
G:
Seed age (h)
20
35
Bioproduction of Conjugated Linoleic Acid in Yogurt
International Journal of Biotechnology for Wellness Industries, 2014, Vol. 3, No. 2
65
a
Table 2: Experimental Plackett-Burman Matrix and Yield of CLA Production
Run
A
B
C
D
E
F
G
CLA yield (g/l)
1
1
-1
-1
1
-1
1
1
7.34 ± 0.5
2
1
1
-1
-1
1
-1
1
6.76 ± 0.6
3
1
1
1
-1
-1
1
-1
0.30 ± 2.2
4
-1
1
1
1
-1
-1
1
3.98 ± 0.5
5
1
-1
1
1
1
-1
-1
2.26 ± 5.5
6
-1
1
-1
1
1
1
-1
0.45 ± 6.7
7
-1
-1
1
-1
1
1
1
9.34 ± 1.9
8
-1
-1
-1
-1
-1
-1
-1
6.39 ± 2.7
a
All data are the average of three replications with standard deviations of the means.
3. RESULTS AND DISCUSSION
mechanism of oxidation of linoleic acid and formation of
linoleic acid radical. It can be interesting to search
whether further increase in the amount of whey powder
may cause more increase in CLA content. This result is
supported by previous report [32, 33] who shows that
addition of skim milk into dairy products led to higher
production of CLA. It may be due to high protein
content in whey powder. Proteins can act as hydrogen
donors and increase isomerisation of linoleic acids in
first step of bio-hydrogenation. As a result, linoleic acid
radicals converted to CLA.
The results of 8 trials conducted by PBD are
summarized in Table 2. The coefficients for the seven
variables were determined and were used for
calculation of predicted yield which are shown in Table
3. Table 3 also gives a comparison of the
experimentally determined citric acid production yield
and productivity to those predicted. Then the student’s
t-test was performed to determine the significance of
each variable employed (t-value =coefficient/Sb). A
significant level of 0.05 was acceptable. Statistical
calculations for PBD of CLA production from date sugar
has summarized in Table 3.
Results showed that in probiotic yogurt containing L.
acidophilus and B. lactis along with traditional yogurt
bacterial culture higher amount of CLA was produced
at 35°C compared to 40 °C. Increase in temperature
may lead suitable condition for growth of L. bulgaricus
as a strong acid producer in process of production of
Based on the results of this study, addition of 4%
whey powder to whole milk increased CLA content.
This increase could be due to proteins role in
Table 3: Statistical Results of Impact of Seven Variables on CLA Production Using Plackett-Burman Design
a
Factors
Effect
Coefficient
t-value
P-value
A (whey)
4.1233
2.0617
6.24
0.000
B (oil)
1.5385
0.7692
2.33
0.033
C (pH)
-1.2650
-0.6325
-1.92
0.074
D (time)
2.8117
1.4058
4.26
0.001
E (Inoculum size)
0.2000
0.1000
0.30
0.766
F (temperature)
-0.9433
-0.2467
-0.75
0.466
G (Inoculum age)
4.5083
2.2542
6.83
0.000
a
Tabulated t-value for degree of freedom 6 and = 0.05 is 2.447.
Table 4: Chemical Characteristics of Yogurt Containing the Highest Amount of CLA
Character
CLA 0.1% w/w
at first
Yogurt containing CLA
9.23
after 1 week
9.18
Acidity 0.01
% w/w
pH
0.76
4.8
Solid lipid
%
11.24
(w/w)
0.03
66
International Journal of Biotechnology for Wellness Industries, 2014, Vol. 3, No. 2
probiotic yogurt. However accumulation of produced
lactic acid had a detrimental impact on CLA. By
increase in temperature form 35°C to 39°C, L.
delbrueckii subspecies bulgaricus in mixed culture had
a more suitable condition as an active acid producer
inhibited the probiotic bacterial growth. Kim et al.,
[2002] also reported by decrease in pH of culture to 4.6
the amount of produced CLA in final product
decreased.
Khosravi-Darani et al.
Inoculum age and size of 36 h and 0.8% lead to
more production of CLA. Bio-hydrogenation is a
process which needs energy to take place, thus, it
seldom occurs in old cells. According to curve obtained
from pH changes in probiotic yogurt, the produced CLA
was higher in logarithmic phase and addition of
substrate rich in fat at pH 6 was the most efficient
factor.
Sensory Evaluation
Therefore, addition of 0.4% of inoculated milk grape
seed oil as a rich source of linoleic acid caused higher
CLA amount. There is a direct relationship between the
degree of conjugated linoleic and linoleic acid in
fermented dairy products. A report by Kim et al. [2002],
supported this hypothesis. This recent study showed
that use of grape seed oil besides using mixed cultures
of probiotic bacteria L. acidophilus and B. lactis along
with traditional bacteria to produce yogurt (S. salivarius
subspecies thermophilus and L. delbrueckii subspecies
bulgaricus) increased the CLA. Kim et al., [22] reported
that the addition of sunflower oil to the valve 2.5% fat
fermented with lactic acid starters including regular
starters of yogurt, 10-6 min right before incubation
increased CLA content in the final product. CLA
produced by cells in logarithmic growth phase were
reported as highest value. Addition of 4% of inoculated
milk Grape seed oil in probiotic yogurt was effective in
CLA production. Lim et al. also showed that by addition
of 0.1% linoleic acid into milk, the amount of CLA
production was increased from 0.71 to 2.95 micro
grams in each gram of non fat yogurt [34]. Addition of
sunflower oil to the milk for production of yogurt
containing S. salivarius and L. delbrueckii subspecies
bulgaricus the amount of CLA increased from 4.8 mg to
7 mg fat in final product. Using a rich resource of
linoleic acid such as grape seed oil which contains
more than 77% linoleic acid in presence of lactic acid
bacteria that are commonly associated with production
of probiotic yogurt along with two probiotic bacteria i.e.
L acidophilus and B. lactis, caused increase in the
amount of produced CLA [32, 33].
The better condition to terminate the incubation is
when the pH of the dairy product reached to 6. In acidic
condition CLA production may be inhibited [35-37]. At
incubation time of 37 h, probiotic bacteria were
survived and their growth related metabolite production
could be developed. Moreover, this can inhibit the
detrimental impact of lactic acids produced by L.
delbrueckii subspecies bulgaricus. It should be noted
production of organic acids in yogurt is directly related
to bacterial growth.
Sensory evaluation was conducted by 30 untrained
panelists on probiotic yogurt with and without grape
seed oil in order to compare the taste, texture and
flavor between them. Samples and control were
presented to panelists, separately. For this test, a non
parametric Kolmogorov-Smirnov method was used and
data were analysed by SPSS software 12.
According to the results taken from this section,
there was no difference between the two samples with
or without grape seed oil in terms of flavor, taste and
texture. Therefore, addition of grape seed oil limited to
the amount used in this study had no any adverse and
unpleasant effect on the flavor, taste and texture of
yogurt samples.
4. CONCLUSION
The best conditions to increase CLA in probiotic
yogurt in present study were as follows: the amount of
added whey powder 4%of the volume of milk
inoculated, incubation temperature 35 °C, the amount
of grape seed oil added 4% of inoculated milk, oil
addition at pH 6, and time of incubation termination 27,
Inoculum age 36 h and size 0.8% respectively.
Concentration of CLA was 188 ppm as measured in
the fat extracted from 2.5mL yogurt dissolved in 5 mL
solvent. Considering the percentage of milk fat used in
this study (3%) produced yogurt contained 0.075 g fat.
Thus in optimum condition, amount of CLA was
3
9.2310 (%w/w) and in control sample it was 7.07
3
10 (%w/w) fat. No much research conducted on this
issue. Many researchers had also considered the
positive role of probiotic bacteria along with traditional
bacteria of yogurt to increase the CLA in yogurt without
fat. During fermentation, the majority of CLA isomers
were in the culture supernatant, but with washed cells
obtained at the early stationary phase, 30 h, about 40%
was detected in the cellular lipid.
Bioproduction of Conjugated Linoleic Acid in Yogurt
International Journal of Biotechnology for Wellness Industries, 2014, Vol. 3, No. 2
Experimental design such as Taguchi [38] and PBD
[39] can be used successfully in biotechnological
research to screen the process variables influencing on
biomass and metabolite production. The second step
will be optimization and response surface methodology.
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Received on 10-06-2014
Accepted on 18-07-2014
Published on 22-07-2014
DOI: http://dx.doi.org/10.6000/1927-3037.2014.03.02.4
© 2014 Khosravi-Darani et al.; Licensee Lifescience Global.
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